The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

The Runx2 transcription factor is required for commitment of mesenchymal cells to bone lineages and is a major regulator of osteoblast-specific gene expression. Runx2 is subject to a number of post-transcriptional controls including selective proteolysis and phosphorylation. We previously reported that Runx2 is phosphorylated and activated by the ERK/MAPK pathway (Xiao, G., Jiang, D., Thomas, P., Benson, M. D., Guan, K., Karsenty, G., and Franceschi, R. T. (2000) J. Biol. Chem. 275, 4453-4459). In this study, we used a combination of in vitro and in vivo phosphorylation analysis, mass spectroscopy, and functional assays to identify two sites at Ser 301 and Ser 319 within the proline/serine/threonine domain of Runx2 that are required for this regulation. These sites are phosphorylated by activated ERK1 in vitro and in cell culture. In addition to confirming ERKdependent phosphorylation at Ser 319 , mass spectroscopy identified two other ERK-phosphorylated sites at Ser 43 and Ser 510 . Furthermore, introduction of S301A,S319A mutations rendered Runx2 resistant to MAPK-dependent activation and reduced its ability to stimulate osteoblast-specific gene expression and differentiation after transfection into Runx2-null calvarial cells and mesenchymal cells. In contrast, S301E,S319E Runx2 mutants had enhanced transcriptional activity that was minimally dependent on MAPK signaling, consistent with the addition of a negative charge mimicking serine phosphorylation. These results emphasize the important role played by Runx2 phosphorylation in the control of osteoblast gene expression and provide a mechanism to explain how physiological signals acting on bone through the ERK/MAPK pathway can stimulate osteoblast-specific gene expression.The bone cell lineage is controlled by a hierarchy of transcription factors that are expressed in a defined temporal sequence. Runx2, an essential factor for both hypertrophic cartilage and bone formation, is expressed very early in skeletal development, first appearing coincident with the formation of mesenchymal condensations (1). Subsequent development of the osteoblast lineage requires at least two additional factors; Osterix, which is essential for subsequent progression of the osteoblast lineage, and ATF4, which regulates osteoblast activity, particularly in postnatal animals (2, 3). Runx2 expression continues during the later stages of bone development and persists in regions of active bone remodeling throughout life. Skeletal development in Runx2-deficient mice fails to progress beyond the cartilage anlage stage, whereas dominant-negative suppression of Runx2 even in postnatal animals inhibits osteoblast activity and bone formation (4). Thus, Runx2 is required for both the initial formation of osteoblasts and hypertrophic chondrocytes during development and for sustained osteoblast differentiation during bone remodeling.Consistent with its multiple roles in bone formation, Runx2 is highly regulated. In addition to transcriptional control by factors such as bone morphogenetic proteins...

(2004) Cell 117, 387-398). However, the mechanisms of ATF4 in bone cells are still not clear. In this study, we determined the molecular mechanisms through which ATF4 activates the mouse osteocalcin (Ocn) gene 2 (mOG2) expression and mOG2 promoter activity. ATF4 increased the levels of Ocn mRNA and mOG2 promoter activity in Runx2-containing osteoblasts but not in non-osteoblastic cells that lack detectable Runx2 protein. However, ATF4 increased Ocn mRNA and mOG2 promoter activity in non-osteoblastic cells when Runx2 was co-expressed. Mutational analysis of the OSE1 (ATF4-binding site) and the two OSE2s (Runx2-binding sites) in the 657-bp mOG2 promoter demonstrated that ATF4 and Runx2 activate Ocn via cooperative interactions with these sites. Pull-down assays using nuclear extracts from osteoblasts or COS-7 cells overexpressing ATF4 and Runx2 showed that both factors are present in either anti-ATF4 and anti-Runx2 immunoprecipitates. In contrast, pull-down assays using purified glutathione S-transferase fusion proteins were unable to demonstrate a direct physical interaction between ATF4 and Runx2. Thus, accessory factors are likely involved in stabilizing interactions between these two molecules. Regions within Runx2 required for ATF4 complex formation and activation were identified. Deletion analysis showed that the leucine zipper domain of ATF4 is critical for Runx2 activation. This study is the first demonstration that cooperative interactions between ATF4 and Runx2/Cbfa1 stimulate osteoblast-specific Ocn expression and suggests that this regulation may represent a novel intramolecular mechanism regulating Runx2 activity and, thereby, osteoblast differentiation and bone formation.

The differentiation of osteoblasts from mesenchymal precursors requires a series of cell fate decisions controlled by a hierarchy of transcription factors. These include RUNX2, Osterix (OSX), ATF4 and a large number of nuclear coregulators. During bone development, initial RUNX2 expression coincides with the formation of mesenchymal condensations and precedes the branching of chondrogenic and osteogenic lineages. Given its central role in bone development, it is not surprising that RUNX2 is subject to a variety of controls. These include posttranslational modification, especially phosphorylation, and interactions with accessory nuclear factors. Specific examples of RUNX2 regulation to be reviewed include phosphorylation by the ERK/MAP kinase pathway and interactions with DLX5. RUNX2 is regulated via phosphorylation of critical serine residues in the proline/serine/threonine domain. In vivo, the transgenic expression of constitutively active MAP kinase in osteoblasts accelerated skeletal development, while a dominant-negative MAPK retarded development in a RUNX2-dependent manner. DLX5-RUNX2 complexes can be detected in osteoblasts and this interaction plays a critical role in maintaining osteoblast-specific expression of the bone sialoprotein gene. These studies allow us to begin understanding the complex mechanisms necessary to fine-tune bone formation as mesenchymal progenitors progress down the osteoblast lineage.